Amylin
Amylin is a 37 amino acid protein hormone. It was isolated, purified and chemically characterized as the major component of amyloid deposits in the islets of pancreases of human Type 2 diabetics (Cooper et al., Proc. Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The amylin molecule has two important post-translational modifications: the C-terminus is amidated, and the cysteines in positions 2 and 7 are cross-linked to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of the Lys-Arg dibasic amino acid proteolytic cleavage signal, prior to the N-terminal codon for Lys, and the Gly prior to the Lys-Arg proteolytic signal at the C-terminal position, a typical sequence for amidation by protein amidating enzyme, PAM (Cooper et al., Biochem. Biophys. Acta, 1014:247-258 (1989)). Amylin is the subject of United Kingdom patent application Serial No. 8709871, filed Apr. 27, 1987, and corresponding U.S. applications filed Apr. 27, 1988, Nov. 23, 1988 and May 2, 1989.
In Type 1 diabetes, amylin has been shown to be deficient and combined replacement with amylin has been proposed as a preferred treatment over insulin alone, for instance in limiting hypoglycemic episodes. The use of amylin for the treatment of diabetes mellitus is the subject of United Kingdom patent application Serial No. 8720115 filed on Aug. 26, 1987, by G. J. S. Cooper, and filed as patent application Ser. No. 236,985 in the United States on Aug. 26, 1988. Pharmaceutical compositions containing amylin and amylin plus insulin are described in U.S. Pat. No. 5,124,314, issued Jun. 23, 1992.
Excess amylin action mimics key features of Type 2 diabetes and amylin blockade has been proposed as a novel therapeutic strategy. It has been disclosed in commonly-owned copending U.S. patent application Ser. No. 275,475, filed Nov. 23, 1988 by Cooper, G. J. S. et al., the contents of which are incorporated herein by reference, that amylin causes reduction in both basal and insulin-stimulated incorporation of labelled glucose into glycogen in skeletal muscle. The latter effect was also disclosed to be shared by CGRP (see also Leighton, B. and Cooper, G. J. S., Nature, 335:632-635 (1988)). Amylin and CGRP were approximately equipotent, showing marked activity at 1 to 10 nM. Amylin is also reported to reduce insulin-stimulated uptake of glucose into skeletal muscle and reduce glycogen content (Young et al., Amer. J. Physiol. 259:457-461 (1990)). The treatment of Type 2 diabetes and insulin resistance with amylin antagonists is disclosed.
Both the chemical structure and the gene sequence amylin have been said to support the determination that it is a biologically active or "messenger" molecule. The chemical structure is nearly 50% identical to the calcitonin-gene-related peptides (CGRP), also 37 amino acid proteins which are widespread neurotransmitters with many potent biological actions, including vasodilation. Amylin and CGRP share the .sup.2 Cys-.sup.7 Cys disulfide bridge and the C-terminal amide, both of which are essential for full biologic activity (Cooper et al. Proc. Natl. Acad. Sci., 85-7763-7766 (1988)).
Amylin may be one member of a family of related peptides which include CGRP, insulin, insulin-like growth factors, and the relaxins and which share common genetic heritage (Cooper, G. J. S., et al., Prog. Growth Factor Research 1:99-105 (1989)). The two peptides calcitonin and CGRP-1 share common parentage in the calcitonin gene where alternative processing of the primary mRNA transcript leads to the generation of the two distinct peptides, which share only limited sequence homology (about 30%) (Amara, S. G. et al., Science, 229:1094-1097 (1985)). The amylin gene sequence is typical for a secreted messenger protein, with the mRNA coding a prepropeptide with processing sites for production of the secreted protein within the Golgi or secretory granules. Amylin is mainly co-localized with insulin in beta cell granules and may share the proteolytic processing enzymes that generate insulin from pro-insulin.
Amylin is primarily synthesized in pancreatic beta cells and is secreted in response to nutrient stimuli such as glucose and arginine. Studies with cloned beta-cell tumor lines (Moore et al., Biochem. Biophys. Res. Commun., 179(1) (1991)), isolated islets (Kanatsuka et al., FEBS Lett., 259(1), 199-201 (1989)) and perfused rat pancreases (Ogawa et al., J. Clin. Invest., 85:973-976 (1990)) have shown that short pulses, 10 to 20 minutes, of nutrient secretagogues such as glucose and arginine, stimulate release of amylin as well as insulin. The molar amylin:insulin ratio of the secreted proteins varies between preparations from about 0.01 to 0.4, but appears not to vary much with different stimuli in any one preparation. However, during prolonged stimulation by elevated glucose, the amylin:insulin ratio can progressively increase (Gedulin et al., Biochem. Biophys. Res. Commun., 180(1):782-789 (1991)). Thus, perhaps because gene expression and rate of translation are independently controlled, amylin and insulin are not always secreted in a constant ratio.
Amylin-like immunoreactivity has been measured in circulating blood in rodents and humans by a variety of radioimmunoassays all of which use rabbit anti-amylin antiserum, and most of which use an extraction and concentration procedure to increase assay sensitivity. In normal humans, fasting amylin levels from 1 to 10 pM and post-prandial or post-glucose levels of 5 to 20 pM have been reported (e.g., Hartter et al., Diabetologia, 34:52-54 (1991)); Sanke et al., Diabetologia, 34:129-132 (1991)); Koda et al., The Lancet, 339:1179-1180 (1992)). In obese, insulin-resistant individuals, post-food amylin levels can go higher, reaching up to about 50 pM. For comparison, the values for fasting and post-prandial insulin are 20 to 50 pM, and 100 to 300 pM respectively in healthy people, with perhaps 3- to 4-fold higher levels in insulin-resistant people. In Type 1 diabetes, where beta-cells are destroyed, amylin levels are at or below the levels of detection and do not rise in response to glucose (Koda et al., The Lancet, 339, 1179-1180 (1992)). In normal mice and rats, basal amylin levels have been reported from 30 to 100 pM, while values up to 600 pM have been measured in certain insulin-resistant, diabetic strains of rodents (e.g., Huang et al., Hypertension, 19:I-101-I-109 (1991)); Gill et al., Life Sciences, 48:703-710 (1991)).
It has been discovered that certain actions of amylin are similar to known non-metabolic actions of CGRP and calcitonin; however, the metabolic actions of amylin discovered during investigations of this newly identified protein appear to reflect its primary biologic role. At least some of these metabolic actions are mimicked by CGRP, albeit at doses which are markedly vasodilatory (see, e.g., Leighton et al., Nature, 335:632-635 (1988)); Molina et al., Diabetes, 39:260-265 (1990)).
The first discovered action of amylin was the reduction of insulin-stimulated incorporation of glucose into glycogen in rat skeletal muscle (Leighton et al., Nature, 335:632-635 (1988)); the muscle was made "insulin-resistant". Subsequent work with rat soleus muscle ex-vivo and in vitro has indicated that amylin reduces glycogen synthase activity, promotes conversion of glycogen phosphorylase from the inactive b form to the active a form, promotes net loss of glycogen (in the presence or absence of insulin), increases glucose-6-phosphate levels, and can increase lactate output (see, e.g., Deems et al., Biochem. Biophys. Res. Commun., 181(1):116-120 (1991)); Young et al., FEBS Letts, 281(1,2):149-151 (1991)). Whether amylin interferes with glucose transport per se is uncertain (see e.g. Young et al., Am. J. Physiol., 259:E457-E461 (1990); Zierath et al., Diabetologia, 35:26-31 (1992)). Studies of amylin and insulin dose-response relations show that amylin acts as a non-competitive or functional antagonist of insulin in skeletal muscle (Young et al., Am. J. Physiol., Am. J. Physiol., 263(2):E274-E281 (1992)). Thus, at an effective concentration of amyrin, no concentration of insulin can overcome amylin action. There is no evidence that amylin interferes with insulin binding to its receptors, or the subsequent activation of insulin receptor tyrosine kinase (Follett et al., Clinical Research 39(1):39A (1991)); Koopmans et al., Diabetologia, 34, 218-224 (1991)). The actions of amylin on skeletal muscle resemble those of adrenaline (epinephrine). However, while adrenaline's actions are believed to be mediated largely by cAMP, some workers have concluded that amylin's actions are not mediated by cAMP (see Deems et al., Biochem. Biophys. Res. Commun., 181(1):116-120 (1991)).
It is believed that amylin acts through receptors present in plasma membranes. It has been reported that amylin works in skeletal muscle via a receptor-mediated mechanism that promotes glycogenolysis, by activating the rate-limiting enzyme for glycogen breakdown, phosphorylase a (Young, A. et al., FEBS Letts, 281:149-151 (1991)). Studies of amylin and CGRP, and the effect of the antagonist .sup.8-37 CGRP, suggest that amylin acts via its own receptor (Wang et al., FEBS Letts., 219:195-198 (1991 b)), counter to the conclusion of other workers that amylin may act primarily at CGRP receptors (e.g., Chantry et al., Biochem. J., 277:139-143 (1991)); Galeazza et al., Peptides, 12:585-591 (1991)); Zhu et al., Biochem. Biophys. Res. Commun., 177(2):771-776 (1991)).
While amylin has marked effects on hepatic fuel metabolism in vivo, there is no general agreement as to what amylin actions are seen in isolated hepatocytes or perfused liver. The available data do not support the idea that amylin promotes hepatic glycogenolysis, i.e., it does not act like glucagon (e.g., Stephens, et al., Diabetes, 40:395-400 (1991)); Gomez-Foix et al., Biochem J., 276:607-610 (1991)). It has been suggested that amylin may act on the liver to promote conversion of lactate to glycogen and to enhance the amount of glucose able to be liberated by glucagon (see Roden et al., Diabetologia, 35:116-120 (1992)). Thus, amylin could act as an anabolic partner to insulin in liver, in contrast to its catabolic action in muscle.
The effect of amylin on regional hemodynamic actions, including renal blood flow, in conscious rats was recently reported (Gardiner et al., Diabetes, 40:948-951 (1991)). The authors noted that infusion of rat amylin was associated with greater renal vasodilation and less mesenteric vasoconstriction than is seen with infusion of human .alpha.-CGRP. They concluded that, by promoting renal hyperemia to a greater extent than did .alpha.-CGRP, rat amylin could cause less marked stimulation of the renin-angiotensin system, and thus, less secondary angiotensin II-mediated vasoconstriction. It was also noted, however, that during coninfusion of human .alpha.-.sup.8-37 CGRP and rat amylin renal and mesenteric vasoconstrictions were unmasked, presumably due to unopposed vasoconstrictor effects of angiotensin II, and that this finding is similar to that seen during coninfusion of human .alpha.-CGRP and human .alpha.-.sup.8-37 CGRP (id. at 951).
In fat cells, contrary to its adrenalin-like action in muscle, amylin has no detectable actions on insulin-stimulated glucose uptake, incorporation of glucose into triglyceride CO.sub.2 production (Cooper et al., Proc. Natl. Acad. Sci., 85:7763-7766 (1988)) epinephrine-stimulated lipolysis, or insulin-inhibition of lipolysis (Lupien and Young, "Diabetes Nutrition and Metabolism--Clinical and Experimental" (in press). Amylin thus exerts tissue-specific effects, with direct action on skeletal muscle, marked indirect (via supply of substrate) and perhaps direct effects on liver, while adipocytes appear "blind" to the presence or absence of amylin. No direct effects of amylin on kidney tissue have been reported.
It has also been reported that amylin can have marked effects on secretion of insulin. In isolated islets (Ohsawa et al., Biochem. Biophys. Res. Commun., 160(2):961-967 (1989)), in the perfused pancreas (Silvestre et al., Reg. Pept., 31-23-31 (1991)), and in the intact rat (Young et al., Mol. Cell. Endocrinol., 84:R1-R5 (1992)), some experiments indicate that amylin down-regulates insulin secretion. The perfused pancreas experiments point to selective down-regulation of the secretory response to glucose with sparing of the response to arginine. Other workers, however, have been unable to detect effects of amylin on isolated .beta.-cells, on isolated islets, or in the whole animal (see Broderick et al., Biochem. Biophys. Res. Comm., Vol. 177:932-938 (1991) and references therein).
The most striking effect of amylin in vivo is stimulation of a sharp rise in plasma lactate, followed by a rise in plasma glucose (Young et al., FEBS Letts, 281(1,2):149-151 (1991)). Evidence indicates that the increased lactate provides substrate for glucose production and that amylin actions can occur independent of changes in insulin or glucagon. In "glucose clamp" experiments, amylin infusions cause "insulin resistance", both by reducing peripheral glucose disposal, and by limiting insulin-mediated suppression of hepatic glucose output (e.g., Frontoni et al., Diabetes, 40:568-573 (1991)); Koopmans et al., Diabetologia, 34, 218-224 (1991)).
In lightly anesthetized rats which were fasted for 18 hours to deplete their stores of hepatic glycogen, amylin injections stimulated rises in plasma lactate from about 0.5 to 1.5 mM followed by a prolonged increase in plasma glucose levels from about 6 to 11 mM. These effects were observed for both intravenous and subcutaneous injections (Young et al., FEBS Letts, 281(1,2):149-151 (1991)). The effects of amylin in fed animals differ quantitatively from its effects in fasted animals. In fed rats, with presumably normal liver glycogen stores, amylin causes a more pronounced and prolonged rise in plasma lactate; however, there is only a modest rise in plasma glucose. It has been suggested that amylin promotes the "return limb" of the Cori cycle, i.e., muscle glycogen via breakdown to lactate provides substrate for hepatic gluconeogenesis and glycogen production and probably triglyceride synthesis. Insulin drives the forward limb, i.e., uptake of glucose into muscle and production of muscle glycogen. Insulin and amylin can thus be seen as partners in regulating the "indirect" pathway of post-prandial hepatic glycogen repletion. "Insulin resistance" in muscle and liver may be under normal, physiologic regulation by amylin.
Non-metabolic actions of amylin include vasodilator effects which may be mediated by interaction with CGRP vascular receptors. Reported in vivo tests suggest that amylin is at least about 100 to 1000 times less potent than CGRP as a vasodilator (Brain et al., Eur. J. Pharmacol., 183:2221 (1990); Wang et al., FEBS Letts., 291:195-198 (1991)). Injected into the brain, amylin has been reported to suppress food intake (e.g., Chance et al., Brain RES., 539, 352-354 (1991)), an action shared with CGRP and calcitonin. The effective concentrations at the cells that mediate this action are not known. Amylin has also been reported to have effects both on isolated osteoclasts where it caused cell quiescence, and in vivo where it was reported to lower plasma calcium by up to 20% in rats, in rabbits, and in humans with Paget's disease ( see, e.g., Zaidi et al., J. Bone Mineral Res., S293 (1990). From the available data, amylin seems to be 10 to 30 times less potent than human calcitonin for these actions. Interestingly, it was reported that amylin appeared to increase osteoclast cAMP production but not to increase cytosolic Ca.sup.2+, while calcitonin does both (Alam et al., Biochem. Biophys. Res. Commun., 179(1):134-139 (1991)). It was suggested, though not established, that calcitonin may act via two receptor types and that amylin may interact with one of these.
Infusing amylin receptor antagonists may be used to alter glucoregulation. .sup.8-37 CGRP is a demonstrated amylin blocker in vitro and in vivo (Wang et al., Biochem. Biophys. Res. Commun., 181(3):1288-1293 (1991)), and was found to alter glucoregulation following an arginine infusion in fed rats (Young et al., Mol. Cell. Endocrino., 84:R1-R5 (1992)). The initial increase in glucose concentration is attributed to arginine-stimulated glucagon secretion from islet alpha cells; the subsequent restoration of basal glucose is attributed to insulin action along with changes in other glucoregulatory hormones. When the action of amylin is blocked by preinfusion of .sup.8-37 hCGRP, the initial glucose increase is not significantly different, but there is a subsequent fall in glucose concentration to well below the basal level, which is restored only after some 80 minutes. Thus, glucoregulation following this challenge with an islet secretagogue was altered by infusion of an amylin receptor antagonist. Additionally, insulin concentrations were measured at half hour intervals and it was found that insulin concentration 30 minutes following the arginine infusion was almost twice as high in animals infused with an amylin receptor antagonist as in the normal controls. .sup.8-37 CGRP is also an effective CGRP antagonist. However, very similar results were seen with another amylin antagonist, AC66, which is selective for amylin receptors compared with CGRP receptors (Young et al., Mol. Cell. Endocrino., 84:R1-R5 (1992)). These results are said to support the conclusion that blockade of amylin action can exert important therapeutic benefits in Type 2 diabetes.
Patients with Type 1 diabetes, in addition to a lack of insulin, are reported to have marked amylin deficiency. As noted above, data show that amylin expression and secretion by pancreatic beta-cells is absent or well below normal in Type 1 diabetes. In several animal models of Type 1 diabetes, amylin secretion and gene expression are depressed (Cooper et al., Diabetes, 497-500 (1991); Ogawa et al., J. Clin. Invest., 85:973-976 (1990)). Measurements of plasma amylin in Type I diabetic patients show that amylin is deficient in these patients after an overnight fast, and that a glucose load does not elicit any increase in amylin levels (Koda et al., The Lancet, 339:1179-1180 (1992)).
Renin
The renin-angiotensin system is an extensively studied physiological control system; among its key functions are the regulation of body fluid and ionic composition, renal function and blood pressure. Excessive or inappropriate activity is well recognized as an important cause of hypertension and a contributor to the problems of heart failure. Methods of inhibition of the renin-angiotensin system have been developed as important treatments for hypertension and heart failure. A summary of the physiology, pharmacology, pathology and clinical aspects of this area is set out in Chapter 27 (page 639-653) of Goodman & Gilman's "The Pharmacological Basis of Therapeutics" (7th Edition, 1985).
Renin is a highly specific aspartyl proteinase of molecular weight about 40,000 Daltons, produced and secreted by juxtaglomerula cells of the kidney. Renin acts on the plasma substrate, angiotensinogen, to split off the non-inactive decapeptide angiotensin I. Angiotensin I is in turn converted to angiotensin II, the major bioactive molecule in this "cascade". Renin in itself has no recognized biological activity beyond its action as a proteolytic enzyme; rather it can be considered to be an endocrine factor, derived from renal tissue. Renal renin release is reported to be stimulated by several mechanisms, including: falls in blood pressure; reduced blood volume; reduced plasma sodium concentration; .beta.-adrenoceptor stimulation by circulating epinephrine or sympathetic nerve activity; and a variety of other bioactive molecules, such as prostaglandins, cytokines, and growth factors whose physiologic and pathologic relevance is less or not at all clear. There is renin production in certain other locations, particularly the brain where the renin-angiotensin system is thought to be a local regulator. The very low levels of plasma renin activity observed after removal of the kidneys indicates that most of circulating renin is of renal origin.
The main biological and medical importance of renin is believed to reside in its ability to generate angiotensins from angiotensinogen. It is currently considered that most of the biologic and pathologic actions of renin are due to the bioactivity of angiotensin II. Other enzymes, widely distributed in the body, are capable of further degrading angiotensin II to angiotensin III and then to inactive peptide fragments. Angiotensin III is not regarded to have important biological activity but rather to be an inactive metabolite of angiotensin II.
The first discovered activity of angiotensin II was a potent ability to increase the blood pressure in intact animals. This effect is now believed to occur by both direct action on blood vessels and indirectly by activation of the sympathetic nervous system. In man, the actions of angiotensin, when directly infused intravenously, are believed to result mainly from a direct action on small blood vessels which in most cases are constricted, thereby increasing the resistance to flow and raising the blood pressure. Angiotensin also acts directly on cardiac muscle cells with a number of consequences including an increase in the force of contraction. The various acute actions of angiotensin on the heart and on the arterial and venous vasculature typically result in a small decrease in cardiac output and because of the constriction, the work of the heart is generally increased.
Angiotensin also has important actions on the adrenal cortex, an endocrine gland an important function of which is the secretion of aldosterone. Angiotensin at very low concentrations directly stimulates the synthesis and secretion of aldosterone. Aldosterone acts on the kidney to enhance the retention of sodium, and retention of sodium, when excessive, is considered an important contributor to hypertension and to the problems of heart failure. Excessive or inappropriate secretion of renin with consequent excess production of angiotensin and consequent excess production of aldosterone can therefore contribute to these pathologic conditions.
Yet another action of angiotensin is on kidney tissue itself where, by action on both intra-renal blood vessels and renal tubules, low concentrations of angiotensin (in the range found in normals subjects and those with hypertension and heart failure) produces an increase of sodium and fluid retention which can contribute to both hypertension and heart failure. It is important to emphasize that concentrations of circulating renin and angiotensin II found in normal individuals, or individuals with essential hypertension fall in the range where there is promotion of aldosterone secretion, action on the kidney, but little or no actual vasoconstriction. Higher levels are required for the vasoconstriction and directly consequent acute blood pressure to occur. But, modestly elevated levels, by the effect on the kidney and body fluid balance, will lead to chronically elevated blood pressure and contribute to the problems of cardiac failure. See, e.g., Chapters 14 and 15 in Hladky & Rink, "Body Fluid and Kidney Physiology" (Edward Arnold, London, 1986).
The concept that excess or inappropriate renin secretion with consequent excess or inappropriate angiotensin formation and action importantly contribute to hypertensive disease and to problems associated with cardiac failure is supported by studies of a number of types of inhibitors of the renin-angiotensin system. Angiotensin converting-enzyme (ACE) inhibitors can slow or block the formation of angiotensin II from angiotensin I. Many drugs of this class have proved to be effective anti-hypertensive agents and are increasingly being employed in the treatment of cardiac failure. Another class of compounds, the angiotensin II receptor antagonists, are presently under clinical development. In both experimental animals and in clinical trials these compounds have shown efficacy in the treatment of hypertension. Others have sought to develop specific inhibitors of renin itself. Compounds of this class have shown efficacy in animal models and in human studies in lowering blood pressure and in treating hypertension.
Each of these classes of compounds is expected to have inhibitory action on the renin-angiotensin system wherever it may be located in the tissues of the body. The available data shows that widespread inhibition of renin or the blockade of angiotensin receptors are effective therapeutic agents. Of the three classes of agents just mentioned, only the ACE inhibitors have been in general practice long enough for the adverse effect profile to have become widely known. Among the adverse effects of these agents are rashes, disturbances of the sense of taste, vertigo, headache, hypotension, and various gastro-intestinal disturbances. Neutropenia has been described as a serious but rare toxicity. Some of these side effects have been attributed to interference with the metabolism of other bioactive molecules termed kinins; however, the evidence does not rule out interference with activity of the renin-angiotensin system in tissues other than the kidney in causing these adverse effects.
Another approach to therapeutic control of the renin-angiotensin system is to reduce the renal secretion of renin. This can be anticipated to remove the majority of circulating renin and markedly reduce angiotensin activity; yet this approach should leave local renin-angiotensin systems, in brain for example, unaffected. At least one current therapeutic approach to hypertension, .beta.-adrenoceptor blockers such as proprandol are thought to work in part by inhibiting renal renin release (Goodman and Gillman, supra at page 195). This conclusion fits with the finding that (1) epinephrine and other .beta.-agonists can stimulate renal renin release and (2) proprandol has been shown to reduce plasma renin levels. However, .beta.-adrenergic agents that reduce renal renin release have significant undesirable adverse effects at effective hypotensive doses, including: cardiac depression, airways constriction, headache, lassitude, depression and gastro-intestinal disturbances. Therefore, the discovery of new agents which control renin release by novel mechanisms and lack the adverse side effects of .beta.-blockers are desirable and important therapeutic targets.
There is also known to be a highly significant linkage between essential hypertension, hyperinsulinemia, and insulin resistance in what has been termed "Syndrome X". It was thought that the high insulin levels were an important cause of the hypertension via an action on the kidney tubules to enhance sodium retention. Newly published evidence, however, is said not to support this concept (Jarrett, R. J., "In defence of insulin: a critique of syndrome X," The Lancet, 340:469-471) Aug. 22, 1992)). The linkage between insulin resistance and hypertension makes the combination of anti-hypertensive activity with the ability to reduce insulin resistance, a very attractive therapy. Indeed, it has been shown that captopril, an important ACE inhibitor, has modest effects in relieving insulin resistance and this was indicated to be therapeutically advantageous. (Watson N. and Sandler M., Curr. Med. Res. Opin., 12(6):374-378 (1991); Kodama J. et al., Diabetes Care, 13(11):1109-11111 (1990); Lithell et al., J. Cardiovasc. Pharmacol., 15 Suppl. 5:S46-S52 (1990)). Thus, a therapeutic modality that acted both to reduce renin levels and to reduce insulin resistance, is a particularly attractive therapeutic target.